In multi-cellular organisms, localization of proteins to particular domains
within a cell is fundamental to the generation of asymmetry during development
and to the polarization of differentiated cells. Apart from protein targeting,
many cell types employ localized translation of mRNAs to ensure restricted accumulation
of proteins. As a model system, my laboratory studies the Drosophila nanos (nos)
mRNA, whose localized translation is essential for patterning of the anterior-posterior
body axis during embryonic development. Remarkably, two post-transcriptional
mechanisms, subcellular mRNA localization and translational control, are coupled
to restrict Nos protein synthesis to the posterior of the embryo. Nos protein
is required in the posterior of the embryo for abdominal development but must
be excluded from the anterior to permit head and thorax development. Localization
of nos RNA to the posterior pole of the embryo is required to activate nos translation;
when localization is abolished, nos mRNA remains translationally repressed.
Localization is not sufficient to restrict nos to the posterior, however; more
than 95% of nos mRNA in the embryo is unlocalized. Translational repression
of this unlocalized mRNA is essential for anterior development. We have shown
that translational repression of unlocalized nos mRNA is mediated by a 90 nucleotide
translational control element (TCE) within the nos 3' untranslated region (3'UTR).

Both the primary sequence and predicted secondary structure of the TCE is
conserved between D. melanogaster and D. virilis and TCE function
is conserved as well. Through a systematic analysis of TCE mutations, we have
shown that TCE function in vivo requires formation of a bipartite structure
consisting of two stem-loops. One RNA binding protein, Smg, has been identified
that interacts with TCE stem-loop II; TCE function requires at least two additional,
as yet unidentified factors, however.

Posterior localization is mediated by a 540 nucleotide cis-acting localization
signal that can be subdivided into partially redundant localization elements,
one of which is coincident with the TCE. Each element contains a domain whose
sequence is conserved between the D. melanogaster and D. virilis
nos 3'UTRs. Results from our analysis of localization element function indicate
that wild-type nos RNA localization requires recognition of multiple sequence
or structural motifs. Our demonstration that the D. virilis nos 3'UTR confers
wild-type localization in a D. melanogaster embryo suggests that the conserved
domains define functionally relevant motifs that are the targets for binding
by putative localization factors. Through biochemical assays, we have identified
several candidate localization factors that interact specifically with these
sequences.